Endothelial Cyp26b1 restrains murine heart valve growth during development.

Endothelial cells (ECs) are critical to proper heart valve development, directly contributing to the mesenchyme of the cardiac cushions, which progressively transform into mature valves. To date, investigators have lacked sufficient markers of valve ECs to evaluate their contributions during valve morphogenesis fully. As a result, it has been unclear whether the well-characterized regional differentiation of valves correlates with any endothelial domains in the heart. Furthermore, it has been difficult to ascertain whether endothelial heterogeneity in the heart influences underlying mesenchymal zones in an angiocrine manner. To identify regionally expressed EC genes in the heart valves, we screened publicly available databases and assembled a toolkit of endothelial-enriched genes. We identified Cyp26b1 as one of many endothelial enriched genes found to be expressed in the endocardium of the developing cushions and valves. Here, we show that Cyp26b1 is required for normal heart valve development. Genetic ablation of Cyp26b1 in mouse embryos leads to abnormally thickened aortic valve leaflets, which is due in part to increased endothelial and mesenchymal cell proliferation in the remodeling valves. In addition, Cyp26b1 mutant hearts display ventricular septal defects (VSDs) in a portion of null embryos. We show that loss of Cyp26b1 results in upregulation of retinoic acid (RA) target genes, supporting the observation that Cyp26b1 has RA-dependent roles. Together, this work identifies a novel role for Cyp26b1 in heart valve morphogenesis and points to a role of RA in this process. Understanding the spatiotemporal expression dynamics of cardiac EC genes will pave the way for investigation of both normal and dysfunctional heart valve development.

Cyp26b1 is an essential regulator of distal airway epithelial differentiation during lung development.

Proper organ development depends on coordinated communication between multiple cell types. Retinoic acid (RA) is an autocrine and paracrine signaling molecule essential for the development of most organs, including the lung. Despite extensive work detailing effects of RA deficiency in early lung morphogenesis, little is known about how RA regulates late gestational lung maturation. Here, we investigate the role of the RA catabolizing protein Cyp26b1 in the lung. Cyp26b1 is highly enriched in lung endothelial cells (ECs) throughout development. We find that loss of Cyp26b1 leads to reduction of alveolar type 1 cells, failure of alveolar inflation and early postnatal lethality in mouse. Furthermore, we observe expansion of distal epithelial progenitors, but no appreciable changes in proximal airways, ECs or stromal populations. Exogenous administration of RA during late gestation partially mimics these defects; however, transcriptional analyses comparing Cyp26b1-/- with RA-treated lungs reveal overlapping, but distinct, responses. These data suggest that defects observed in Cyp26b1-/- lungs are caused by both RA-dependent and RA-independent mechanisms. This work reports crucial cellular crosstalk during lung development involving Cyp26b1-expressing endothelium and identifies a novel RA modulator in lung development.

Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis.

Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochrome P450 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.

A variant in CYP26B1 associated with esophageal squamous cell carcinoma risk by affecting retinoic acid metabolism.

All-trans retinoic acid (ATRA) is the natural and synthetic analogue of vitamin A, playing an essential tumor suppressive role in multiple cancers including the esophageal squamous cell carcinoma (ESCC). Cytochrome P450 family 26 subfamily B member 1 (CYP26B1) exerts a critical regulator of ATRA levels through specific inactivation of ATRA to hydroxylated forms. Our previous exome-wide analyses revealed a rare missense variant in CYP26B1 significantly associated with ESCC risk in the Chinese population. However, it is still unclear whether there are common variants in CYP26B1 affect the susceptibility of ESCC and the tumor promotion role of CYP26B1 in vivo. In this research, we conducted a two-stage case-control study comprised of 5057 ESCC cases and 5397 controls, followed by a series of biochemical experiments to explore the function of CYP26B1 and its common variants in the tumorigenesis of ESCC. Intriguingly, we identified a missense variant rs2241057[A>G] in the fourth exon of CYP26B1 significantly associated with the ESCC risk (combined odds ratio = 1.28; 95% confidence interval = 1.15-1.42; p = 2.96 × 10-6 ). Through further functional analysis, we demonstrated that ESCC cells with the overexpression of rs2241057[G] had a significant lower level of retinoic acid, compared with the overexpression of rs2241057[A] or the control vector. In addition, the CYP26B1 overexpression and knock-out ESCC cells affected cell proliferation rate both in vitro and in vivo. These results highlighted the carcinogenicity of CYP26B1 related to the ATRA metabolism in ESCC risk.

A Functional Polymorphism Downstream of Vitamin A Regulator Gene CYP26B1 Is Associated with Hand Osteoarthritis.

While genetic analyses have revealed ~100 risk loci associated with osteoarthritis (OA), only eight have been linked to hand OA. Besides, these studies were performed in predominantly European and Caucasian ancestries. Here, we conducted a genome-wide association study in the Han Chinese population to identify genetic variations associated with the disease. We recruited a total of 1136 individuals (n = 420 hand OA-affected; n = 716 unaffected control subjects) of Han Chinese ancestry. We carried out genotyping using Axiom Asia Precisi on Medicine Research Array, and we employed the RegulomeDB database and RoadMap DNase I Hypersensitivity Sites annotations to further narrow down our potential candidate variants. Genetic variants identified were tested in the Geisinger's hand OA cohort selected from the Geisinger MyCode community health initiative (MyCode®). We also performed a luciferase reporter assay to confirm the potential impact of top candidate single-nucleotide polymorphisms (SNPs) on hand OA. We identified six associated SNPs (p-value = 6.76 × 10-7-7.31 × 10-6) clustered at 2p13.2 downstream of the CYP26B1 gene. The strongest association signal identified was rs883313 (p-value = 6.76 × 10-7, odds ratio (OR) = 1.76), followed by rs12713768 (p-value = 1.36 × 10-6, OR = 1.74), near or within the enhancer region closest to the CYP26B1 gene. Our findings showed that the major risk-conferring CC haplotype of SNPs rs12713768 and rs10208040 [strong linkage disequilibrium (LD); D' = 1, r2 = 0.651] drives 18.9% of enhancer expression activity. Our findings highlight that the SNP rs12713768 is associated with susceptibility to and severity of hand OA in the Han Chinese population and that the suggested retinoic acid signaling pathway may play an important role in its pathogenesis.

Oxytocin receptor behavioral effects and cell types in the bed nucleus of the stria terminalis.

Oxytocin is a neuropeptide that can produce anxiolytic effects and promote social approach. However, emerging evidence shows that under some conditions, oxytocin can instead induce anxiety-related behaviors. These diverse effects of oxytocin appear to be mediated by circuit-specific actions. Recent data showed that inhibition of oxytocin receptors (OTRs) in the bed nucleus of the stria terminalis (BNST) was sufficient to increase social approach and decrease social vigilance in female California mice (Peromyscus californicus) exposed to social defeat stress. As a member of the G-protein coupled receptor family, OTRs can induce distinct downstream pathways by coupling to different G-protein isoforms. We show that infusion of carbetocin, a biased OTR-Gq agonist, in the BNST reduced social approach in both female and male California mice. In both females and males, carbetocin also increased social vigilance. To gain insight into cell types that could be mediating this effect, we analyzed previously published single-cell RNAseq data from the BNST and nucleus accumbens (NAc). In the NAc, we and others showed that OTR activation promotes social approach behaviors. In the BNST, Oxtr was expressed in over 40 cell types, that span both posterior and anterior subregions of the BNST. The majority of Oxtr-expressing neurons were GABAergic. In the anterior regions of BNST targeted in our carbetocin experiments, Cyp26b1-expressing neurons had high average Oxtr expression. In the NAc, most Oxtr+ cells were D1 dopamine receptor-expressing neurons and interneurons. These differences in Oxtr cell type distribution may help explain how activation of OTR in BNST versus NAc can have different effects on social approach and social vigilance.

Nonlethal presentations of CYP26B1-related skeletal anomalies and multiple synostoses syndrome.

Retinoic acid exposures as well as defects in the retinoic acid-degrading enzyme CYP26B1 have teratogenic effects on both limb and craniofacial skeleton. An initial report of four individuals described a syndrome of fetal and infantile lethality with craniosynostosis and skeletal anomalies caused by homozygous pathogenic missense variants in CYP26B1. In contrast, a 22-year-old female was reported with a homozygous missense pathogenic variant in CYP26B1 with complex multisuture craniosynostosis and intellectual disability, suggesting that in some cases, biallelic pathogenic variants of CYP26B1 may be compatible with life. Here we describe four additional living individuals from two families with compound heterozygous pathogenic missense variants in CYP26B1. Structural assessment of these additional missense variants places them further from the catalytic site and supports a model consistent with milder nonlethal disease. In addition to previously reported findings of multisuture craniosynostosis, conductive hearing loss, joint contractures, long slender fingers, camptodactly, broad fingertips, and developmental delay/intellectual disability, skeletal imaging in our cases also revealed gracile long bones, gracile ribs, radioulnar synostosis, and carpal and/or tarsal fusions. These individuals broaden the phenotypic range of biallelic pathogenic variants in CYPB26B1 and most significantly clarify that mortality can range from perinatal lethality to survival into adulthood.

CYP26B1 declines postnatally in Sertoli cells independently of androgen action in the mouse testis.

Meiosis begins at puberty and relies on several factors, including androgens and retinoic acid in the mouse testis. CYP26B1 degrades retinoic acid in the testis during prenatal development preventing meiosis initiation. Given the concurrence of meiotic entry and completion of Sertoli cell maturation in response to androgens at puberty in the mouse, we proposed that CYP26B1 is downregulated by androgens in the Sertoli cell during this period. By immunohistochemistry, we showed that CYP26B1 declines in Sertoli cells after birth. However, luciferase reporter assays and quantitative reverse transcription-polymerase chain reaction performed in the prepubertal mouse Sertoli cell line SMAT1 revealed no changes in Cyp26b1 expression in response to androgen treatment. Furthermore, studies carried out using primary Sertoli cells of 10-day-old mice showed no changes in either Cyp26b1 or CYP26B1 expression in response to androgen treatment. In summary, the hereby reported decline in CYP26B1 expression in Sertoli cells towards pubertal onset does not appear to be caused by a direct inhibitory effect of androgens on Sertoli cells in the mouse.

A Schwann cell-enriched circular RNA circ-Ankib1 regulates Schwann cell proliferation following peripheral nerve injury.

Schwann cells (SCs) play an essential role in nerve injury repair. A striking feature of the cellular response to peripheral nerve injury is the proliferation of SCs. Circular (circ)RNAs are enriched in the nervous system and are involved in physiologic and pathologic processes. However, the potential role of circRNAs in SC proliferation post nerve injury remains largely unknown. Using a sciatic nerve crush model, we obtained an expression profiling of circRNAs in injured sciatic nerves in rats by RNA sequencing and bioinformatics analysis, and we further identified a circRNA [circ-ankyrin repeat and in-between Ring finger (IBR) domain containing 1 (Ankib1)] involved in SC proliferation by the transfection of specific small interfering RNAs. Overexpression of circ-Ankib1, which was specifically and highly enriched in SCs, impaired SC proliferation and axon regeneration following sciatic nerve injury. Mechanistically, increased expression of DEx/H-box helicase 9 (DHX9) postinjury might contribute to the down-regulation of circ-Ankib1, which further suppressed cytochrome P450, family 26, subfamily B, polypeptide 1 expression by sponging miR-423-5p, miR-485-5p, and miR-666-3p, leading to the induction of SC proliferation and nerve regeneration. Taken together, our results reveal a crucial role for circRNAs in regulating proliferation of SCs involved in sciatic nerve regeneration; as such, circRNAs may serve as a potential therapeutic avenue for nerve injury repair.-Mao, S., Zhang, S., Zhou, S., Huang, T., Feng, W., Gu, X., Yu, B. A Schwann cell-enriched circular RNA circ-Ankib1 regulates Schwann cell proliferation following peripheral nerve injury.

Isoflavones Suppress Cyp26b1 Expression in the Murine Colonic Lamina Propria.

Isoflavones have many biological activities and are major bioactive components of kakkonto, a traditional Japanese herbal medicine. We previously reported that the combined therapy of oral immune therapy (OIT) and kakkonto downregulates the mRNA expression of Cyp26b1, a major retinoic acid (RA)-degrading enzyme, in the colon of food allergy mice and thereby ameliorates allergic symptoms. In this study, we evaluated the effects of various isoflavones on Cyp26b1 expression in primary cultured lamina propria (LP) cells isolated from the mouse colon. The mRNA expression of Cyp26b1 was extremely downregulated by all isoflavones tested in the LP cells except for puerarin. In particular, genistein and genistin markedly suppressed Cyp26b1 mRNA expression without affecting RA-synthesizing enzyme expression. Moreover, to evaluate the effects of isoflavones on allergic reactions, genistein and genistin were administered to ovalbumin (OVA)-induced food allergy mice. Oral administration of genistin suppressed the development of allergic symptoms. These results raise the possibility that isoflavones elevated the level of RA in the colon by inhibiting RA degradation and then the high concentration of RA in the colon might exert immunosuppressive and antiallergic effects on food allergy mice.

Early inhibition of endothelial retinoid uptake upon myocardial infarction restores cardiac function and prevents cell, tissue, and animal death.

Physiologically, following myocardial infarction (MI), retinoid levels elevate locally in the infarcted area. Whereas therapeutic systemic application of retinoids was shown to reduce the progression of ventricular dilatation and the onset of heart failure, the role of acute physiologically increased retinoids in the infarction zone is unknown to date. To reveal the role of local retinoids in the MI zone is the central aim of this study. Using human cell culture and co-culture models for hypoxia as well as various assays systems, lentivirus-based transgene expression, in silico molecular docking studies, and an MI model in rats, we analysed the impact of the retinoid all-trans retinoic acid (ATRA) on cell signalling, cell viability, tissue survival, heart function, and MI-induced death in rats. Based on our results, ATRA-mediated signalling does aggravate the MI phenotype (e.g. 2.5-fold increased mortality compared to control), whereas 5'-methoxyleoligin (5ML), a new agent which interferes with ATRA-signalling rescues the ATRA-dependent phenotype. On the molecular level, ATRA signalling causes induction of TXNIP, a potent inhibitor of the physiological antioxidant thioredoxin (TRX1) and sensitizes cells to necrotic cell death upon hypoxia. 5ML-mediated prevention of ATRA effects were shown to be based on the inhibition of cellular ATRA uptake by interference with the cholesterol (and retinol) binding motif of the transmembrane protein STRA6. 5ML-mediated inhibition of ATRA uptake led to a strong reduction of ATRA-dependent gene expression, reduced ROS formation, and protection from necrotic cell death. As 5ML exerted a cardioprotective effect, also independent of its inhibition of cellular ATRA uptake, the agent likely has another cardioprotective property, which may rely on the induction of TRX1 activity. In summary, this is the first study to show i) that local retinoids in the early MI zone may worsen disease outcome, ii) that inhibition of endothelial retinoid uptake using 5ML may constitute a novel treatment strategy, and iii) that targeting endothelial and myocardial retinoid uptake (e.g. via STRA6 inhibition) may constitute a novel treatment target in acute MI.

Meiotic initiation in chicken germ cells is regulated by Cyp26b1 and mesonephros.

Our knowledge of mechanisms involved in the meiosis of chicken germ cells is very limited. In mammalian fetal ovaries, the onset of meiosis is dependent on retinoic acid and subsequent upregulation of the Stra8 gene. To clarify the mechanism of meiotic initiation in chicken germ cells, we investigated the role of Cyp26b1, a retinoic acid-degrading enzyme. The Cyp26b1-inhibitor, ketoconazole was used to treat the ex vivo-cultured stage 36 gonads/mesonephroi. Then, the progression of meiosis was studied by histological and immunohistochemical analysis and the level of the transcript for Stra8 was evaluated by a quantitative reverse transcription-polymerase chain reaction in individual ketoconazole-treated gonads after 6 days in culture. The results revealed that meiosis was induced in both testes and right ovary upon inhibition of Cyp26b1 in the ex vivo-cultured gonads, despite downregulation of Stra8 messenger RNA in the treated gonads. Also, meiosis was observed only when mesonephros was cultured alongside the left ovary. These findings demonstrate that in chicken, Stra8 is not the only factor for the entrance into meiosis, and Cyp26b1 and mesonephros play critical regulatory roles for the sex-specific timing of meiotic initiation in birds.

Genetic contribution of retinoid-related genes to neural tube defects.

Rare variants are considered underlying causes of complex diseases. The complex and severe group of disorders called neural tube defects (NTDs) results from failure of the neural tube to close during early embryogenesis. Neural tube closure requires the coordination of numerous signaling pathways, including the precise regulation of retinoic acid (RA) concentration, which is controlled by enzymes involved in RA synthesis and degradation. Here, we used a case-control mutation screen study to reveal rare variants in retinoid-related genes in a Han Chinese NTD population by sequencing six genes in 355 NTD cases and 225 controls. More specific rare variants were found in exonic and upstream regions in NTD cases. The RA-responsive genes CYP26A1, CRABP1, and ALDH1A2 harbored NTD-specific rare variants in their upstream regions. Unexpectedly, the majority of missense variants in NTD cases were found in CYP26B1, which encodes a RA degradation enzyme, whereas no missense variants in this gene were found in controls. Functional analysis indicated that the CYP26B1 NTD variants were inefficient in the degradation of RA using assays of RA-induced transcription and RA-initiated neuronal differentiation. Our study supports the contribution of rare variants in RA-related genes to the etiology of human NTDs.

Osteoblast de- and redifferentiation are controlled by a dynamic response to retinoic acid during zebrafish fin regeneration.

Zebrafish restore amputated fins by forming tissue-specific blastema cells that coordinately regenerate the lost structures. Fin amputation triggers the synthesis of several diffusible signaling factors that are required for regeneration, raising the question of how cell lineage-specific programs are protected from regenerative crosstalk between neighboring fin tissues. During fin regeneration, osteoblasts revert from a non-cycling, mature state to a cycling, preosteoblastic state to establish a pool of progenitors within the blastema. After several rounds of proliferation, preosteoblasts redifferentiate to produce new bone. Blastema formation and proliferation are driven by the continued synthesis of retinoic acid (RA). Here, we find that osteoblast dedifferentiation and redifferentiation are inhibited by RA signaling, and we uncover how the bone regenerative program is achieved against a background of massive RA synthesis. Stump osteoblasts manage to contribute to the blastema by upregulating expression of the RA-degrading enzyme cyp26b1. Redifferentiation is controlled by a presumptive gradient of RA, in which high RA levels towards the distal tip of the blastema suppress redifferentiation. We show that this might be achieved through a mechanism involving repression of Bmp signaling and promotion of Wnt/ß-catenin signaling. In turn, cyp26b1(+) fibroblast-derived blastema cells in the more proximal regenerate serve as a sink to reduce RA levels, thereby allowing differentiation of neighboring preosteoblasts. Our findings reveal a mechanism explaining how the osteoblast regenerative program is protected from adverse crosstalk with neighboring fibroblasts that advances our understanding of the regulation of bone repair by RA.

Mucopolysaccharidosis IIIB and mild skeletal anomalies: coexistence of NAGLU and CYP26B1 missense variations in the same patient in a Chinese family.

BACKGROUND: Sanfilippo type B syndrome (mucopolysac-charidosis type IIIB; MPS IIIB) is an autosomal recessive lysosomal storage disorder. It is caused by a critically reduced α-2-acetamido-2-deoxy-D-glucoside acetamidodeoxy glucohydrolase (α-N-acetylglucosaminidase or NAGLU) activity. Recently, an autosomal recessive disorder of skeletal dysplasia associated with CYP26B1 was reported in three families, in which the patients were all homozygous variations. However, the co-occurrence of two rare diseases in a person is very rare. Here, we reported one patient with two novel pathogenic missense variations in NAGLU and CYP26B1. CASE PRESENTATION: We found an infant with biallelic variation both in NAGLU-compound heterozygous c.1843C > T (p. R615C) and c.1224C > A (p. H408Q) as well as in CYP26B1-compound heterozygous c.529G > A (p. E177K) and c.525C > A (p. H175Q). All variations were novel but predicted pathogenicity according to American College of Medical Genetics and Genomics (ACMG) guidelines. The main phenotypes of the infant were quite different from those previously reported, and some were combinations of the two rare diseases, including epilepsy, early onset epileptic encephalopathy, hypermyotonia, skull deformity, dilatation of the lateral ventricles and premature closure of fontanel. His NAGLU enzyme activity was significantly decreased. CONCLUSIONS: NAGLU and CYP26B1 mutations were related to MPS IIIB and skeletal dysplasia, respectively. Here, we first reported the pathogenic mutations of two genes concurrent in one patient, which not only expands the phenotype and genotype spectra of NAGLU and CYP26B1, but more importantly indicates the possibility of simultaneous occurrence of two rare diseases in one patient. This interesting finding should be attributed to the use of whole exome sequencing (WES), which indicates that we should be aware of the importance of WES in diagnosing rare diseases.

Human foetal ovary shares meiotic preventing factors with the developing testis.

STUDY QUESTION: How can pre-meiotic germ cells persist in the human foetal ovary? SUMMARY ANSWER: Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis. WHAT IS KNOWN ALREADY: The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive. STUDY DESIGN SIZE, DURATION: In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization. PARTICIPANTS/MATERIALS SETTING, METHODS: Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures. MAIN RESULTS AND THE ROLE OF CHANCE: Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells. LARGE SCALE DATA: N/A. LIMITATIONS REASON FOR CAUTION: Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis. WIDER IMPLICATIONS OF THE FINDINGS: We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Université Paris Diderot-Paris 7 and Université Paris-Sud, CEA, INSERM, and Agence de la Biomédecine. The authors declare no conflict of interest.

Control of retinoid levels by CYP26B1 is important for lymphatic vascular development in the mouse embryo.

During embryogenesis, lymphatic endothelial progenitor cells first arise from a subset of blood vascular endothelial cells in the dorsolateral aspects of the cardinal veins. The molecular cues responsible for defining the regionalisation of such a discrete pool of progenitors remain uncharacterised. Here we identify a novel function for CYP26B1, an enzyme known to play a role in tissue morphogenesis by fine-tuning retinoic acid (RA) concentration, in regulating lymphangiogenesis. Cyp26b1-null mice, in which RA levels are elevated, exhibited an increased number of lymphatic endothelial progenitor cells in the cardinal veins, together with hyperplastic, blood filled lymph sacs and hyperplastic dermal lymphatic vessels. Conversely, mice over-expressing Cyp26b1 had hypoplastic lymph sacs and lymphatic vessels. Our data suggest that RA clearance by CYP26B1 in the vicinity of lymphatic endothelial progenitor cells is important for determining the position and size of the progenitor pool specified. Our studies identify a genetic pathway that underpins the architecture of the developing lymphatics and define CYP26B1 as a novel modulator of lymphatic vascular patterning.

CYP26B1 promotes male germ cell differentiation by suppressing STRA8-dependent meiotic and STRA8-independent mitotic pathways.

Germ cell sex is defined by factors derived from somatic cells. CYP26B1 is known to be a male sex-promoting factor that inactivates retinoic acid (RA) in somatic cells. In CYP26B1-null XY gonads, germ cells are exposed to a higher level of RA than in normal XY gonads and this activates Stra8 to induce meiosis while male-specific gene expression is suppressed. However, it is unknown whether meiotic entry by an elevated level of RA is responsible for the suppression of male-type gene expression. To address this question, we have generated Cyp26b1/Stra8 double knockout (dKO) embryos. We successfully suppressed the induction of meiosis in CYP26B1-null XY germ cells by removing the Stra8 gene. Concomitantly, we found that the male genetic program represented by the expression of NANOS2 and DNMT3L was totally rescued in about half of dKO germ cells, indicating that meiotic entry causes the suppression of male differentiation. However, half of the germ cells still failed to enter the appropriate male pathway in the dKO condition. Using microarray analyses together with immunohistochemistry, we found that KIT expression was accompanied by mitotic activation, but was canceled by inhibition of the RA signaling pathway. Taken together, we conclude that inhibition of RA is one of the essential factors to promote male germ cell differentiation, and that CYP26B1 suppresses two distinct genetic programs induced by RA: a Stra8-dependent meiotic pathway, and a Stra8-independent mitotic pathway.

Cyp26b1 within the growth plate regulates bone growth in juvenile mice.

Retinoic acid (RA) is an active metabolite of vitamin A and plays important roles in embryonic development. CYP26 enzymes degrade RA and have specific expression patterns that produce a RA gradient, which regulates the patterning of various structures in the embryo. However, it has not been addressed whether a RA gradient also exists and functions in organs after birth. We found localized RA activities in the diaphyseal portion of the growth plate cartilage were associated with the specific expression of Cyp26b1 in the epiphyseal portion in juvenile mice. To disturb the distribution of RA, we generated mice lacking Cyp26b1 specifically in chondrocytes (Cyp26b1(Δchon) cKO). These mice showed reduced skeletal growth in the juvenile stage. Additionally, their growth plate cartilage showed decreased proliferation rates of proliferative chondrocytes, which was associated with a reduced height in the zone of proliferative chondrocytes, and closed focally by four weeks of age, while wild-type mouse growth plates never closed. Feeding the Cyp26b1 cKO mice a vitamin A-deficient diet partially reversed these abnormalities of the growth plate cartilage. These results collectively suggest that Cyp26b1 in the growth plate regulates the proliferation rates of chondrocytes and is responsible for the normal function of the growth plate and growing bones in juvenile mice, probably by limiting the RA distribution in the growth plate proliferating zone.

Utility and limits of Hprt-Cre technology in generating mutant mouse embryos.

BACKGROUND: Hprt-Cre doubles the prevalence of homozygous null embryos per litter versus heterozygous breedings without decreasing litter size. Resulting mutant embryos are genotypically and phenotypically equivalent between strategies. We set out to confirm the effectiveness of this approach with other alleles and hypothesized that it would increase efficiency in generating compound mutants. MATERIALS AND METHODS: Null mutants for Cyp26b1, Pitx2, and Shh were generated with Hprt-Cre from conditional alleles as were double and triple allelic combinations of Fgfr2IIIb, Raldh2, and Cyp26b1. Embryos were genotyped and phenotyped by whole mount photography, histology, and immunohistochemistry. RESULTS: Fifty percent of Hprt-Cre litters were homozygous null for Cyp26b1 (15/29) and Pitx2 (75/143), with phenotypic and genotypic equivalence to mutants from standard heterozygous breedings. In multi-allele breedings, mutant embryos constituted half of litters without significant embryo loss. In contrast, Shh breedings yielded a smaller ratio of embryos carrying two recombined alleles (6 of 16), with a significant litter size reduction because of early embryonic lethality (16 live embryos from 38 deciduae). CONCLUSIONS: Hprt-Cre can be used to efficiently generate large numbers of mutant embryos with a number of alleles. Compound mutant generation was equally efficient. However, efficiency is reduced for genes whose protein product potentially interacts with the Hprt pathway (e.g., Shh).